The rewiring of transcriptional circuits has been proposed as a major source of evolutionary novelty. This proposal seeks to determine the detailed molecular mechanisms that underlie transcriptional rewiring using unicellular yeasts as a model system. The strategy is based on direct experimentation in three yeasts-- Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida albicans- using gene knockout collections, genome wide transcriptional profiling and chromatin immunoprecipitation. Circuit comparisons among these three yeasts will reveal specific examples of transcriptional rewiring and provide hypotheses for the molecular mechanisms by which the wiring changes occurred. Specific hypotheses will be tested and refined by phylogenetic comparisons of all sequenced ascomycete genomes (18 currently) and additional experimentation in modern yeasts. Prior and ongoing work on the evolution of the well-characterized yeast mating-type circuitry will serve as a guide for these studies. A cell carefully regulates the transcription of each one of its many genes. In humans, abnormalities in this complex process (whether inherited or acquired) can lead to many diseases, including a variety of cancers. Transcriptional circuits are the product of billions of years of evolution, and a complete understanding of them must include a consideration of how they arose and how they can change. Plasticity in transcriptional circuitry is well-documented, and a deeper understanding of it will provide insights into recognizing and perhaps treating cases where it leads to aberrant physiologies.